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Ca 2؉ /calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of cellular Ca 2؉ signaling. Several inhibitors are commonly used to study CaMKII function, but these inhibitors all lack specificity. CaM-KIIN is a natural, specific CaMKII inhibitor protein. CN21 (derived from CaM-KIIN amino acids 43-63) showed full specificity and potency of CaMKII inhibition. CNs completely blocked Ca 2؉ -stimulated and autonomous substrate phosphorylation by CaMKII and autophosphorylation at T305. However, T286 autophosphorylation (the autophosphorylation generating autonomous activity) was only mildly affected. Two mechanisms can explain this unusual differential inhibitor effect. First, CNs inhibited activity by interacting with the CaMKII T-site (and thereby also interfered with NMDA-type glutamate receptor binding to the T-site). Because of this, the CaMKII region surrounding T286 competed with CNs for T-site interaction, whereas other substrates did not. Second, the intersubunit T286 autophosphorylation requires CaM binding both to the "kinase" and the "substrate" subunit. CNs dramatically decreased CaM dissociation, thus facilitating the ability of CaM to make T286 accessible for phosphorylation. Tat-fusion made CN21 cell penetrating, as demonstrated by a strong inhibition of filopodia motility in neurons and insulin secrection from isolated Langerhans' islets. These results reveal the inhibitory mechanism of CaM-KIIN and establish a powerful new tool for dissecting CaMKII function. INTRODUCTIONCa 2ϩ /calmodulin-dependent protein kinase II (CaMKII) is a multifunctional protein kinase best known for its critical role in learning and memory (for review, see Lisman and McIntyre, 2001;Soderling et al., 2001;Hudmon and Schulman, 2002;Lisman et al., 2002). CaMKII is highly expressed in the brain (Erondu and Kennedy, 1985), but at least one of its four isoforms (␣, ␤, ␥, and ␦) has been found in every cell type examined (Tobimatsu and Fujisawa, 1989;Bayer et al., 1999;Tombes et al., 2003). Numerous cellular functions of CaMKII have been described previously, both in and outside the nervous system. These include regulation of various ion channels (Worrell and Frizzell, 1991;Wang and Best, 1992;Roeper et al., 1997;Derkach et al., 1999;Dzhura et al., 2000), gene expression (Nghiem et al., 1994;Ramirez et al., 1997;Meffert et al., 2003), cell cycle/proliferation control (Baitinger et al., 1990;Patel et al., 1999;Matsumoto and Maller, 2002;Illario et al., 2003), apoptotic and excitotoxic cell death (Laabich and Cooper, 2000;Fladmark et al., 2002), cell morphology (Wu and Cline, 1998;Fink et al., 2003), and filopodia motility (Fink et al., 2003). CaMKII also has been implicated in regulation of insulin secretion (for review, see Easom, 1999); however, this conclusion is largely based on experiments using KN inhibitors, which also affect the Ca 2ϩ channels required for secretion (see below).CaMKII forms multimeric holoenzymes (Bennett et al., 1983;Kanaseki et al., 1991;Kolodziej et al., 2000;Morris and Torok, 2001;Hoel...

SUMMARY Traditionally, hippocampal long-term potentiation (LTP) of synaptic strength requires Ca2+/calmodulin(CaM)-dependent protein kinase II (CaMKII) and other kinases, while long-term depression (LTD) requires phosphatases. Here we found that LTD also requires CaMKII and its phospho-T286-induced “autonomous” (Ca2+-independent) activity. However, while LTP is known to induce phosphorylation of the AMPA-type glutamate receptor (AMPAR) subunit GluA1 at S831, LTD instead induced CaMKII-mediated phosphorylation at S567, a site known to reduce synaptic GluA1 localization. GluA1 S831 phosphorylation by “autonomous” CaMKII was further stimulated by Ca2+/CaM, as expected for traditional substrates. By contrast, GluA1 S567 represents a distinct substrate-class that is unaffected by such stimulation. This differential regulation caused GluA1 S831 to be favored by LTP-type stimuli (strong but brief), while GluA1 S567 was favored by LTD-type stimuli (weak but prolonged). Thus, requirement of autonomous CaMKII in opposing forms of plasticity involves distinct substrate classes that are differentially regulated to enable stimulus-dependent substrate-site preference.

Ca 2؉ /calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of physiological glutamate signaling involved in higher brain functions. Here, we show CaMKII involvement in pathological glutamate signaling relevant in stroke. The novel inhibitor tatCN21 was neuroprotective even when added hours after glutamate insults. By contrast, the "traditional" inhibitor KN93 attenuated excitotoxicity only when present during the insult. Both inhibitors efficiently blocked Ca 2؉ /CaM-stimulated CaMKII activity, CaMKII interaction with NR2B and aggregation of CaMKII holoenzymes. However, only tatCN21 but not KN93 blocked the Ca 2؉ -independent "autonomous" activity generated by Thr-286 autophosphorylation, the hallmark feature of CaMKII regulation. Mutational analysis further validated autonomous CaMKII activity as the drug target crucial for post-insult neuroprotection. Overexpression of CaMKII wild type but not the autonomy-deficient T286A mutant significantly increased glutamate-induced neuronal death. Maybe most importantly, tatCN21 also significantly reduced infarct size in a mouse stroke model (middle cerebral arterial occlusion) when injected (1 mg/kg intravenously) 1 h after onset of arterial occlusion. Together, these data demonstrate that inhibition of autonomous CaMKII activity provides a promising therapeutic avenue for post-insult neuro-protection after stroke.Glutamate is the most abundant excitatory neurotransmitter in the mammalian brain. However, excessive glutamate release causes Ca 2ϩ -dependent excitotoxic neuronal death in pathological situations such as focal cerebral ischemia (stroke) (for review see Refs. 1-4). Most glutamate receptors are involved in excitotoxicity, but especially important are the Ca 2ϩ -permeable ionotropic receptors such as the NMDA 4 -type glutamate receptor (NMDAR) (5-8). Extensive attempts to develop a stroke therapy by targeting glutamate receptors have resulted in disappointment (for review see Refs. 9, 10), suggesting that alternative strategies will be necessary. Currently, the only approved pharmacological treatment of stroke patients is hemolytic therapy with tissue plasminogen activator. However, less than 2% of patients actually receive tissue plasminogen activator. Although tissue plasminogen activator is effective in stroke caused by blood clots, it is actually contraindicated in hemorrhagic stroke, and diagnostic evaluation pushes most patients beyond the therapeutically effective time window (11-13).The Ca 2ϩ /calmodulin (Ca 2ϩ /CaM)-dependent protein kinase II (CaMKII) is a major physiological downstream target of glutamate-induced Ca 2ϩ signaling (for review see Refs. 14 -17) and was examined in this study for involvement in pathological excitotoxic glutamate signaling. CaMKII is highly expressed in brain where it participates in NMDAR-dependent long term potentiation and learning and memory (14 -17). CaMKII forms multimeric holoenzymes, and each kinase subunit is activated separately by Ca 2ϩ /CaM. An inter-subunit autophosphorylation at Thr-286 ...

The Ca 2؉ /calmodulin (CaM)-dependent protein kinase II (CaMKII)␤ has morphogenic functions in neurons not shared by the ␣ isoform. CaMKII␤ contains three exons (v1, v3, and v4) not present in the CaMKII␣ gene, and two of these exons (v1 and v4) are subject to differential alternative splicing. We show here that CaMKII␤, but not ␣, mediated bundling of F-actin filaments in vitro. Most importantly, inclusion of exon v1 was required for CaMKII␤ association with the F-actin cytoskeleton within cells. CaMKII␤e, which is the dominant variant around birth and lacks exon v1 sequences, failed to associate with F-actin. By contrast, CaMKII␤, which instead lacks exon v4, associated with F-actin as full-length CaMKII␤. Previous studies with CaMKII␤ mutants have indicated a role of nonstimulated kinase activity in enhancing dendritic arborization. Here, we show that F-actin-targeted CaMKII␤, but not ␣, was able to phosphorylate actin in vitro even by nonstimulated basal activity in absence of Ca 2؉ /CaM. In rat pancreatic islets and in skeletal muscle, the actin-associated CaMKII␤ and ␤M were the predominant variants, respectively. Thus, cytoskeletal targeting may mediate functions of CaMKII␤ variants also outside the nervous system. INTRODUCTIONChanges in synaptic connectivity between neurons are widely thought to underlie higher brain functions such as learning and memory and are also important during development. Long-lasting changes in connectivity are often associated with morphological plasticity in structure or number of synaptic connections (for reviews, see Huntley et al., 2002;McGee and Bredt, 2003;Lamprecht and LeDoux, 2004;Segal, 2005). The ␣ isoform of Ca 2ϩ /calmodulin(CaM)-dependent protein kinase II (CaMKII) has been studied extensively because of its prominent role in regulating the strength of individual synaptic connections (for examples, see Malinow et al., 1989;Silva et al., 1992;Giese et al., 1998; for reviews, see Malenka and Nicoll, 1999;Lisman et al., 2002;Griffith, 2004). Much less is known about the other major brain isoform, CaMKII␤. However, CaMKII␤ has specific morphogenic functions in regulating dendritic arborization and synapse density not shared by the ␣ isoform (Fink et al., 2003). This isoform specificity is thought to be mediated by the specific binding of CaMKII␤, but not ␣ to F-actin (Shen et al., 1998;Fink et al., 2003).The four CaMKII isoforms encoded by different genes (␣, ␤, ␥, and ␦) are highly homologous to each other and can phosphorylate and regulate a variety of substrate proteins in response to Ca 2ϩ signals (for reviews, see Soderling et al., 2001;Hudmon and Schulman, 2002;Lisman et al., 2002;Colbran and Brown, 2004). The multimeric CaMKII holoenzymes are composed of 12 subunits (Kolodziej et al., 2000;Morris and Torok, 2001;Rosenberg et al., 2005;Rosenberg et al., 2006) of a single isoform or combinations of different isoforms (Bayer et al., 1998;Shen et al., 1998;Brocke et al., 1999;Lantsman and Tombes, 2005). All CaMKII isoforms contain an N-terminal kinase domain and a ...

CHD4 (chromodomain helicase DNA-binding protein 4) ATPase is a major subunit of the repressive NuRD (nucleosome remodelling and deacetylase) complex, which is involved in transcriptional regulation and development. CHD4 contains two PHD (plant homeodomain) fingers of unknown function. Here we show that the second PHD finger (PHD2) of CHD4 recognizes the N-terminus of histone H3 and that this interaction is facilitated by acetylation or methylation of Lys9 (H3K9ac and H3K9me respectively) but is inhibited by methylation of Lys4 (H3K4me) or acetylation of Ala1 (H3A1ac). An 18 μM binding affinity toward unmodified H3 rises to 0.6 μM for H3K9ac and to 0.9 μM for H3K9me3, whereas it drops to 2.0 mM for H3K4me3, as measured by tryptophan fluorescence and NMR. A peptide library screen further shows that phosphorylation of Thr3,Thr6 or Ser10 abolishes this interaction. A model of the PHD2–H3 complex, generated using a combination of NMR, data-driven docking and mutagenesis data, reveals an elongated site on the PHD2 surface where the H3 peptide is bound. Together our findings suggest that the PHD2 finger plays a role in targeting of the CHD4/NuRD complex to chromatin.

The Ca 2؉ /calmodulin (CaM)-dependent protein kinase II (CaMKII) and the NMDA-type glutamate receptor are key regulators of synaptic plasticity underlying learning and memory. Direct binding of CaMKII to the NMDA receptor subunit GluN2B (formerly known as NR2B) (i) is induced by Ca 2؉ /CaM but outlasts this initial Ca 2؉ -stimulus, (ii) mediates CaMKII translocation to synapses, and (iii) regulates synaptic strength. CaMKII binds to GluN2B around S1303, the major CaMKII phosphorylation site on GluN2B. We show here that a phosphomimetic S1303D mutation inhibited CaM-induced CaMKII binding to GluN2B in vitro, presenting a conundrum how binding can occur within cells, where high ATP concentration should promote S1303 phosphorylation. Surprisingly, addition of ATP actually enhanced the binding. Mutational analysis revealed that this positive net effect was caused by four modulatory effects of ATP, two positive (direct nucleotide binding and CaMKII T286 autophosphorylation) and two negative (GluN2B S1303 phosphorylation and CaMKII T305/6 autophosphorylation). Imaging showed positive regulation by nucleotide binding also within transfected HEK cells and neurons. In fact, nucleotide binding was a requirement for efficient CaMKII interaction with GluN2B in cells, while T286 autophosphorylation was not. Kinetic considerations support a model in which positive regulation by nucleotide binding and T286 autophosphorylation occurs faster than negative modulation by GluN2B S1303 and CaMKII T305/6 phosphorylation, allowing efficient CaMKII binding to GluN2B despite the inhibitory effects of the two slower reactions.The Ca 2ϩ

Outside of Fragile X syndrome (FXS), the role of Fragile-X Mental Retardation Protein (FMRP) in mediating neuropsychological abnormalities is not clear. FMRP, p70-S6 kinase (S6K) and protein phosphatase 2A (PP2A) are thought to cooperate as a dynamic signaling complex. In our prior work, adult rats have enhanced CA1 hippocampal long-term depression (LTD) following an early life seizure (ELS). We now show that mGluR-mediated LTD (mLTD) is specifically enhanced following ELS, similar to FMRP knock-outs. Total FMRP expression is unchanged but S6K is hyperphosphorylated, consistent with S6K overactivation. We postulated that either disruption of the FMRP-S6K-PP2A complex and/or removal of this complex from synapses could explain our findings. Using subcellular fractionation, we were surprised to find that concentrations of FMRP and PP2A were undisturbed in the synaptosomal compartment but reduced in parallel in the cytosolic compartment. Following ELS FMRP phosphorylation was reduced in the cytosolic compartment and increased in the synaptic compartment, in parallel with the compartmentalization of S6K activation. Furthermore, FMRP and PP2A remain bound following ELS. In contrast, the interaction of S6K with FMRP is reduced by ELS. Blockade of PP2A results in enhanced mLTD; this is occluded by ELS. This suggests a critical role for the location and function of the FMRP-S6K-PP2A signaling complex in limiting the amount of mLTD. Specifically, non-synaptic targeting and the function of the complex may influence the “set-point” for regulating mLTD. Consistent with this, striatal-enriched protein tyrosine phosphatase (STEP), an FMRP “target” which regulates mLTD expression, is specifically increased in the synaptosomal compartment following ELS. Further, we provide behavioral data to suggest that FMRP complex dysfunction may underlie altered socialization, a symptom associated and observed in other rodent models of autism, including FXS.